Differences in Ketanserin Binding in the Ventromedial Hypothalamus of Ewes Responsive or Refractory to Short Days

Abstract

Participation of central 5HT receptors in the inhibition of LH pulsatility during refractoriness to short days (SD) in ewes has been suggested by previous in vivo studies using various 5HT-antagonist such as ketanserin. In the present study, binding of [³H]ketanserin in ewe brain sections was similar to that described in the brain of other species and could correspond with an interaction at 5HT2 receptors sites. Rosenthal analysis from the caudate nucleus was linear (Kd = 3 nM). The displacement studies from the cortex slices showed that the 5HT antagonists such as methysergide, ketanserin, cyproheptadine and spiperone competed with the labelled ligand at nanomolar concentrations whereas serotonin was less active. However, the first 3 drugs recognized different populations of binding sites. Prazosin, an α₁-adrenergic antagonist was inactive, but a slight inhibition of [³H]ketanserin binding was induced by pyrilamine, an H₁ histaminic antagonist, within a nanomolar range. Methysergide (10^–6M), which does not bind to H₁ receptors, was therefore used to determine the nonspecific binding. Quantitative analysis of the binding of 3 nM [³H]ketanserin on sections of the ewe brain at the preopticohypothalamic level was then carried out by autoradiography. The highest binding densities were observed in the caudate nuclei (64.0 fmol/mg tissue Eq) and the mammillary bodies (52.7 fmol/mg tissue Eq) whereas intermediate or low densities were found in the other structures. The anatomical distribution of the labelling was similar to that described in other species for 5HT₂ receptors. Ketanserin binding in these areas was compared between two groups of ovariectomized estradiol-treated Ile-de-France ewes, submitted to artificial short days (SD: 8L: 16D), one group with a high LH pulsatility (responsive to SD) and the other one with a low LH pulsatility (photorefractory to SD). Binding densities were similar for each one of the studied regions between the two groups, except in the ventrolateral part of the mediobasal hypothalamus, where ewes exhibiting high LH pulsatility had a more than 2-fold higher binding density than those with a low LH pulsatility (mean ± SEM, 14.6 ± 1.4 vs. 5.7 ± 1.0 fmol/mg tissue Eq, respectively; p < 0.0016). These results suggest that [³H]ketanserin binding sites in the ventromedial part of the mediobasal hypothalamus could be associated to the regulation of the photoperiodic inhibition of LH at the time of establishment of refractoriness to short days in the Ile-de-France ewe.

Sheep 5HT2A receptors: partial cloning of the coding sequence and mRNA localization by in situ hybridization in the ewe hypothalamus

“…The caudate nucleus was chosen as a positive control tissue since both the 3 H-ketanserin binding in sheep (Le Corre et al 1994) and 5HT2-R mRNA expression in the rat (Pompeiano et al 1994;Wright et al 1995) were found to be high. The present data fit with these results since the ISH labelling was found to be markedly concentrated in this structure.…”

“…Labelled cells were recorded for the whole sections but were counted inside a square of 4×4 mm on each side of the third ventricle, an area which fitted with the maximum of 3 H-ketanserin binding previously found in the premammillary hypothalamus (Le Corre et al 1994). Results are expressed as the mean of the number of labelled cells within the 16-mm 2 square on each side of the ventricle (Fig.…”

“…Studies using antagonists relatively specific to different subtypes of 5HT-R have suggested that 5HT2-R may be involved in the inhibition of LH pulsatility in ovariectomized oestradiol-treated ewes (Le Corre and Chemineau 1993b). In such a model, the determination of brain-binding sites of the tritiated 5HT2 antagonist ketanserin has led to the identification of a specific ventral hypothalamic zone in which the binding density was parallel to a change in LH pulsatility (Le Corre et al 1994).…”

The effect of nutrition on the neural mechanisms potentially involved in melatonin-stimulated LH secretion in female Mediterranean goats

“…In sheep, serotonin receptors are thought to be involved in the neural mechanisms that regulate the release of LH associated with the photoperiod. In this species, the determination of the brain-binding sites of the tritiated 5-hydroxytryptamine receptor 2 (5HT 2 ) antagonist ketanserin led to the identification of a specific ventral hypothalamic zone in which the binding density parallels the change in LH pulsatility (Le Corre et al 1994). In goats, it has recently been shown that inhibitory pathways, such as the endogenous opioid pathways, regulate LH secretion during the early inhibition of LH secretion by long days and during LH stimulation by short days.…”

Melatonin and the seasonal control of reproduction

“…Serotonin inhibits LH pulsatile secretion during seasonal anoestrus, and not during the breeding season [33,50]. This inhibitory effect of serotonin is mediated by 5-HT2 A receptors [34] and interestingly a photoperiod-induced change in the density of 5-HT2 A receptors in the ventrolateral posterior hypothalamus was described [35,54]. 4 [28,42,70].…”